Bioluminescent imaging of Cdk2 inhibition in vivo

Guo-Jun Zhang, Michal Safran, Wenyi Wei, Erik Sorensen, Peter Lassota, Nikolai Zhelev, Donna S. Neuberg, Geoffrey Shapiro, William G. Kaelin Jr

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Abstract

Many proteins and pathways of pharmaceutical interest impinge on ubiquitin ligases or their substrates. The cyclin-dependent kinase (Cdk) inhibitor p27, for example, is polyubiquitylated in a cell cycle−dependent manner by a ubiquitin ligase complex containing the F-box protein Skp2. Regulated turnover of p27 is due, at least partly, to its phosphorylation by Cdk2 on threonine 187, which generates a Skp2-binding site. We made a p27-luciferase (p27Luc) fusion protein and show here that its abundance, like that of p27, is regulated by Skp2 in a cell cycle−dependent manner. As predicted, p27Luc levels increased after blocking Cdk2 activity with inhibitory proteins, peptides or small interfering RNA (siRNA). Accumulation of p27Luc in response to Cdk2 inhibitory drugs (flavopiridol and R-roscovitine) was demonstrable in human tumor cells in vivo using noninvasive bioluminescent imaging. In theory, the approach described here could be used to develop bioluminescent reporters for any drug target that directly or indirectly affects the turnover of a ubiquitin ligase substrate.
Original languageEnglish
Pages (from-to)643-648
Number of pages6
JournalNature Medicine
Volume10
Issue number6
DOIs
StatePublished - May 2004

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Ligases
Ubiquitin
Luciferases
Proteins
Castration
Cell Cycle
Adenofibroma
Substrates
F-Box Proteins
Cyclin-Dependent Kinase Inhibitor p27
Threonine
Small Interfering RNA
Binding Sites
Phosphorylation
Peptides
Neoplasms
Paranasal Sinus Neoplasms
Contraception Behavior
Fusion reactions

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Zhang, G-J., Safran, M., Wei, W., Sorensen, E., Lassota, P., Zhelev, N., ... Kaelin Jr, W. G. (2004). Bioluminescent imaging of Cdk2 inhibition in vivo. Nature Medicine, 10(6), 643-648. DOI: 10.1038/nm1047

Zhang, Guo-Jun; Safran, Michal; Wei, Wenyi; Sorensen, Erik; Lassota, Peter; Zhelev, Nikolai; Neuberg, Donna S.; Shapiro, Geoffrey; Kaelin Jr, William G. / Bioluminescent imaging of Cdk2 inhibition in vivo.

In: Nature Medicine, Vol. 10, No. 6, 05.2004, p. 643-648.

Research output: Contribution to journalArticle

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abstract = "Many proteins and pathways of pharmaceutical interest impinge on ubiquitin ligases or their substrates. The cyclin-dependent kinase (Cdk) inhibitor p27, for example, is polyubiquitylated in a cell cycle−dependent manner by a ubiquitin ligase complex containing the F-box protein Skp2. Regulated turnover of p27 is due, at least partly, to its phosphorylation by Cdk2 on threonine 187, which generates a Skp2-binding site. We made a p27-luciferase (p27Luc) fusion protein and show here that its abundance, like that of p27, is regulated by Skp2 in a cell cycle−dependent manner. As predicted, p27Luc levels increased after blocking Cdk2 activity with inhibitory proteins, peptides or small interfering RNA (siRNA). Accumulation of p27Luc in response to Cdk2 inhibitory drugs (flavopiridol and R-roscovitine) was demonstrable in human tumor cells in vivo using noninvasive bioluminescent imaging. In theory, the approach described here could be used to develop bioluminescent reporters for any drug target that directly or indirectly affects the turnover of a ubiquitin ligase substrate.",
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Zhang, G-J, Safran, M, Wei, W, Sorensen, E, Lassota, P, Zhelev, N, Neuberg, DS, Shapiro, G & Kaelin Jr, WG 2004, 'Bioluminescent imaging of Cdk2 inhibition in vivo' Nature Medicine, vol 10, no. 6, pp. 643-648. DOI: 10.1038/nm1047

Bioluminescent imaging of Cdk2 inhibition in vivo. / Zhang, Guo-Jun; Safran, Michal; Wei, Wenyi; Sorensen, Erik; Lassota, Peter; Zhelev, Nikolai; Neuberg, Donna S.; Shapiro, Geoffrey; Kaelin Jr, William G.

In: Nature Medicine, Vol. 10, No. 6, 05.2004, p. 643-648.

Research output: Contribution to journalArticle

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AB - Many proteins and pathways of pharmaceutical interest impinge on ubiquitin ligases or their substrates. The cyclin-dependent kinase (Cdk) inhibitor p27, for example, is polyubiquitylated in a cell cycle−dependent manner by a ubiquitin ligase complex containing the F-box protein Skp2. Regulated turnover of p27 is due, at least partly, to its phosphorylation by Cdk2 on threonine 187, which generates a Skp2-binding site. We made a p27-luciferase (p27Luc) fusion protein and show here that its abundance, like that of p27, is regulated by Skp2 in a cell cycle−dependent manner. As predicted, p27Luc levels increased after blocking Cdk2 activity with inhibitory proteins, peptides or small interfering RNA (siRNA). Accumulation of p27Luc in response to Cdk2 inhibitory drugs (flavopiridol and R-roscovitine) was demonstrable in human tumor cells in vivo using noninvasive bioluminescent imaging. In theory, the approach described here could be used to develop bioluminescent reporters for any drug target that directly or indirectly affects the turnover of a ubiquitin ligase substrate.

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Zhang G-J, Safran M, Wei W, Sorensen E, Lassota P, Zhelev N et al. Bioluminescent imaging of Cdk2 inhibition in vivo. Nature Medicine. 2004 May;10(6):643-648. Available from, DOI: 10.1038/nm1047